Spectrophotometric assay of pioglitazone hydrochloride using permanganate in acidic and basic media

Article history: Received December 22, 2017 Received in revised form March 21, 2018 Accepted March 25, 2018 Available online March 25, 2018 Pioglitazone hydrochloride (PGH) is an oral anti-hyperglycemic agent used in the treatment of type-2 diabetes mellitus. Potassium permanganate was found to oxidize PGH both in acidic and basic conditions, based on which two simple and sensitive methods were developed for its determination in bulk sample and tablets, and validated. In the first method (indirect method), PGH was reacted with a measured excess of standard permanganate in H2SO4 medium, and the residual oxidant was determined by measuring its absorbance at 550 nm. The second method (Direct method) entails treating PGH with permanganate in NaOH medium, followed by the measurement of the resulting bluish-green manganite at 610 nm. Experimental variables affecting the reactions were studied and optimized. Under optimum conditions, linear relationships with good correlation coefficients were found between absorbance and concentration in the ranges, 1.25 – 25 μg mL-1 (Indirect method) and 1-12 μg mL-1 (Direct method) with respective molar absorptivity values of 1.10 × 104 and 2.77 × 104 l mol-1 cm-1. The limits of detection (LOD) and quantification (LOQ) were 0.36 and 1.08 (Indirect method) and 0.23 and 0.69 μg mL-1 (Direct method). Intra-day and inter-day precisions were satisfactory, with %RSD values of ≤2.11, and the respective accuracies were excellent with %RE values of ≤2. The methods were also validated for robustness, ruggedness and selectivity. The methods were applied to the determination of PGH in its tablets with good accuracy and precision, and no interference from the tablet additives was encountered. The results were also compared with those obtained by a reference method. © 2018 Growing Science Ltd. All rights reserved.

In the presence of reducing agent, potassium permanganate gets reduced to different oxidation states in both acidic and basic media.In acidic solution, manganese(VII) is reduced to manganese(II), whereas in basic medium, it gets reduced to manganese(VI) as shown below: 48 MnO4 -+ 8H + + 5e -= Mn 2+ + 4H2O MnO4 -+ e -= MnO4 2- 0][51][52][53][54][55][56][57][58][59][60] In spite of its extensive application in pharmaceutical analysis, permanganate, as per the literature, has not been used for the determination of PGH.In this work, permanganate was used as an oxidimetric agent for developing two spectrophotometric methods.In the Indirect method, the residual permanganate was measured at 550 nm, after allowing the reaction between PGH and known amount of oxidant in H2SO4 medium.Whereas in the direct method, the bluish-green color of manganite, the product of reaction between drug and permanganate in alkaline medium, was measured at 610 nm, which served as the basis of the Direct method.The methods were found to be much simpler and more sensitive than the existing spectrophotometric methods.

Results and discussion
The proposed methods are based on the redox reaction between permanganate and PGH in acid (Indirect method) or in basic (Direct method) medium.In Indirect method, a known excess of standard KMnO4 was added to PGH in acid medium followed by the determination of the residual oxidant by measuring its absorbance at 550 nm.The decrease in absorbance at 550 nm with respect to water blank was taken as the measure of PGH concentration.In Direct method, K2MnO4 resulting from the reduction of KMnO4 by PGH in alkaline medium was measured at 610 nm and related to PGH concentration.The possible reaction pathways and basis of assays are given in Scheme 1.Preliminary experiments were performed to determine the permanganate concentration which would give a reasonable maximum absorbance at 550 nm in H2SO4 medium; and this was found to be 60 μg mL -1 .Hence, different concentrations of PGH were reacted with 1 mL of 600 μg mL -1 KMnO4 in acid medium, and after the elapsed contact time, the absorbance of the residual permanganate was measured and related to PGH concentration.When a fixed concentration of KMnO4 (60 μg mL -1 ) was reacted with varying concentrations of PGH, the former was consumed in proportion to PGH concentration and there occurred a concomitant fall in the concentration of KMnO4 as shown by the decreasing absorbance values at 550 nm with increase in the PGH concentration.This is depicted in Fig. 2.This facilitated the evaluation of the linear range over which the method could be applicable to the determination of PGH.With hydrochloric acid, there is the likelihood of the reaction taking place and some permanganate may be consumed in the formation of chlorine. 61Hence, the reaction of the oxidant with the drug was carried out in H2SO4 medium.Experiments were performed with 0.5-3.0mL of 2 M H2SO4 and it was found that constant and reproducible absorbance readings were obtained in the range studied.Hence, 2 mL of 2M H2SO4 in a total volume of 10 mL was fixed as the optimum.The redox reaction with 10 μg mL -1 PGH was complete in 15 min, and the absorbance of residual oxidant remained constant for the next 45 min at room temperature (28±2 ᴼC).

Direct method
This method is based on the reduction of permanganate to manganite by PGH in the presence of NaOH, the bluish-green colored chromogen 62 having the absorption maximum at 610 nm (Fig. 3).The formation of the colored product and the sensitivity of the reaction were found to be influenced by the alkali and permanganate concentrations.Maximum and constant absorbance readings were observed with 1 mL of 0.5M NaOH in a total volume of 10 mL (Fig. 4a).The reaction took 10 min for completion, and the bluish-green manganite color was stable for 40 min thereafter (Fig 4b).When a separate experiment was conducted to study the effect of permanganate concentration, it was found that maximum absorbance associated with a minimum blank reading was obtained when 1 mL of 0.1% KMnO4 in a total volume of 10 mL was used.Higher concentrations of permanganate resulted in increased sensitivity, but the blank absorbance showed an increasing trend simultaneously.

Method validation 2.2.1 Linearity, sensitivity, limits of detection and quantification
A linear correlation was found between absorbance at λmax and concentration of PGH (Fig. 5).The slope (m), intercept (b) and correlation coefficient (r) for each system were evaluated using the method of least squares.Optical characteristics such as Beer's law limits, molar absorptivity and Sandell sensitivity values are presented in Table 1.The limits of detection (LOD) and quantitation (LOQ) are also calculated according to ICH guidelines 63 and these data are presented in Table 1.

Precision and accuracy
To check the precision and accuracy of the proposed methods, the assays described under "general procedures" were repeated seven times within the day (intra-day precision and accuracy) and five times on five different days (inter-day precision and accuracy).These assays were performed at three levels of analyte.The RSD values were ≤2.06% (intra-day) and ≤ 2.11% (inter-day) indicating high precision of the methods.%RE values of ≤ 2.0% demonstrate the fair accuracy of the proposed methods.The results of this study are summarized in Table 2.

Robustness and ruggedness
The robustness of the methods was evaluated by making small incremental changes in the volume of reagent and reaction time and the effects of the changes were studied.To determine ruggedness, analyses were performed by three different analysts, and also with three different cuvettes by a single analyst.The intermediate precision, expressed as percent RSD, which is a measure of robustness and ruggedness, was within the acceptable limits as shown in the Table 3, reflecting the robustness and ruggedness of the methods.

Selectivity
There was hardly any change in the absorbance of permanganate when reacted with placebo blank (Indirect method) and no discernible colour developed in direct method.The percentage recoveries of PGH from synthetic mixture solution were 99.67±1.94for Indirect method and 98.72±1.45 for Direct method.This unequivocally demonstrated the non-interference of the inactive ingredients in the assay of PGH.

Application to tablets
The proposed methods were applied to the quantification of PGH in commercial tablets.The results were compared with those of reference method. 5The reference method involve analytical procedure for measurement of absorbance of PGH at 269 nm in 0.1 M HCl. of The accuracy and precision of the proposed methods were further evaluated by applying Student's t-test and variance ratio F-test, respectively.The t-and F-values at 95% confidence level did not exceed the tabulated values and this further confirms that there is no significant difference between the reference method and proposed methods with respect to accuracy and precision.The results of this study are presented in Table 4.

Accuracy assessment by recovery study
Recovery test was performed by applying the standard-addition procedure.The test was done by spiking the pre-analyzed tablet powder with pure PGH at three different levels (50, 100 and 150%) of the content present in the tablet powder (taken) and the total was determined by the proposed methods.
Each test was repeated three times.The recovery percentage values ranged between 98.56 and 103.1% with standard deviation in the range 0.98-2.01%reflecting the high accuracy as well as selectivity of the methods.The results are shown in Table 5.

Conclusion
Reaction of pioglitazone with permanganate in acid and basic media was successfully exploited for the spectrophotometric determination of drug.The reactions provide relatively simple and rapid means of assay of pivoglitazone and its tablet dosage form.The proposed methods are more sensitive than the presently available methods, and have wider linear dynamic ranges.

Acknowledgement
Authors thank Glenmark Pharmaceuticals, Mumbai, India, for gifting pioglitazone pure sample.Prof. K. Basavaiah gratefully acknowledges the financial assistance by UGC, New Delhi, India, in the form of BSR Faculty fellowship.

Apparatus
A Systronics model 106 digital spectrophotometer (Systronics, Ahmedabad, Gujarat, India) equipped with 1-cm matched quartz cells was used for absorbance measurements.

Materials and reagents
All chemicals used were of analytical reagent grade and double distilled water was used throughout the study.
An approximately 0.01M solution of potassium permanganate was prepared by dissolving 0.395 g of the chemical (Merck Ltd., Mumbai, India) in water, the solution was boiled for 10 min to remove any residual manganese(IV) ions, cooled, filtered using glass wool and diluted to 250 mL, and standardized using H.A Bright's procedure. 64This solution was diluted stepwise to get 600 μg mL -1 for Indirect method and 0.1% for Direct method.Sulphuric acid (0.1M and 2M) were prepared by appropriately diluting required volumes of concentrated acid (S.D. Fine Chem., Mumbai, India, sp.gr.1.84) with water to get the required concentrations.A 0.5 M solution of sodium hydroxide were prepared by dissolving about 10.0 g NaOH (Merck, Mumbai, India) in 500 mL of water and standardized against pure potassium hydrogenphthalate. 65This was diluted to get 0.1M with water.

Standard drug solution
Pure sample of PGH was kindly supplied by Glenmark Pharmaceuticals, Mumbai, India, as gift.A 500 μg mL -1 stock standard solution was prepared by dissolving 50 mg of PGH in 0.1M H2SO4 and made up to 100 mL with same acid in a calibrated flask.It was diluted to 50 μg mL -1 PGH for use in Indirect method.Forty mg of PGH were weighed accurately and dissolved in 0.1M NaOH in a 100 mL calibrated flask and diluted to the mark with the same solvent.The solution was then diluted with water to get a working concentration of 40 μg mL -1 for use in Direct method.

. Indirect Method
Different aliquots (0.25-5.0 mL, 50 μg mL -1 ) of PGH solution were transferred into a series of 10 mL standard flasks by means of micro burette and the total volume was adjusted to 5.0 mL with 0.1M H2SO4.Two mL of 2M H2SO4 were added to each flask followed by 1 mL of 600 μg mL -1 KMnO4 solution.The flasks were kept aside for 15 min with occasional shaking before diluting to the mark with water.The absorbance was recorded at 550 nm against a water blank.

Direct Method
Into a series of 10 mL volumetric flasks, 0.25-3.0mL of 40 μg mL -1 PGH solution were buretted and the total volume was made up to 3.0 mL with 0.1M NaOH.To each flask was added 1 mL of 0.5M NaOH followed by 1 mL of 0.1% KMnO4 solution.The flasks were kept aside for 10 min with occasional shaking and the volume was made upto the mark with water.The absorbance was recorded at 610 nm against the reagent blank.
Calibration graphs were prepared by plotting either decreasing absorbance values in Indirect method or increasing absorbance values in Direct method versus concentration of PGH.The unknown concentration was computed from the regression equation, derived using Beer's law data.

Procedure for tablets
A quantity of tablet powder containing 25 mg of PGH was accurately weighed into a 50 mL calibrated flask, added 30 mL of 0.1M H2SO4 and shaken for 20 min.Then, the volume was diluted to the mark with 0.1M H2SO4, mixed and filtered using a Whatman No. 42 filter paper.First 10 mL of the filtrate were discarded and a suitable aliquot of the subsequent portion (500 μg mL -1 in PGH) was diluted with 0.1M H2SO4 to obtain 50 μg mL -1 solution and the analysis was completed following the procedure described earlier by taking 3 mL aliquot in five replicates (Indirect method).In Direct method, a portion of the tablet powder equivalent to 20 mg of PGH was accurately weighed into a 100 mL beaker.The powder was extracted with three 10 mL portions of chloroform and each time the extract was filtered with a Whatman No. 42 filter paper.The filtrate was collected in 100 mL calibrated flask, chloroform was evaporated over a water bath and the residue was dissolved in 0.1M sodium hydroxide and diluted to the mark in a 100 mL calibrated flask with same solvent.The solution was diluted to 40 µg mL -1 and assayed by taking 2 mL aliquot (n = 5).

Procedure for placebo blank and synthetic mixture
A placebo blank containing starch (10 mg), acacia (15 mg), hydroxyl cellulose (10 mg), sodium citrate (10 mg), talc (20 mg), magnesium stearate (15 mg) and sodium alginate (10 mg) was prepared by homogeneous mixing.A 20 mg portion was weighed and solution prepared as described under 'procedure for tablets' and then subjected to analysis.
A synthetic mixture was separately prepared by mixing 50 mg of pure PGH with 40 mg of placebo.Portions containing 25 mg or 20 mg of PGH were separately taken and solutions were prepared as described under "procedure for tablets" and analyzed following the general procedures, and the percentage recovery of PGH was computed.

Fig. 2 .
Fig. 2. Absorption spectra of KMnO4 (60 μg mL -1 ) after reacting with PGH (μg mL -1 ): a) 0.0, b) 5.0, c) 10.0, d) 17.5 and e) 20.0 Sulphuric acid is the most suitable acid, since it has no action upon permanganate in dilute solution.With hydrochloric acid, there is the likelihood of the reaction taking place and some permanganate may be consumed in the formation of chlorine.61Hence, the reaction of the oxidant with the drug was carried out in H2SO4 medium.Experiments were performed with 0.5-3.0mL of 2 M H2SO4 and it was found that constant and reproducible absorbance readings were obtained in the range studied.Hence, 2 mL of 2M H2SO4 in a total volume of 10 mL was fixed as the optimum.The redox reaction with 10 μg mL -1 PGH was complete in 15 min, and the absorbance of residual oxidant remained constant for the next 45 min at room temperature (28±2 ᴼC).

Table 2 .
Results of accuracy and precision study RE-Relative error, RSD-Relative standard deviation

Table 3 .
Results of robustness and ruggedness study expressed as intermediate precision (%RSD) *Reaction times were 13, 15 & 17 min in Indirect method and 8, 10 & 12 min in Direct method.**In Indirect method, H2SO4 volumes were 1.8, 2.0 and 2.2 mL, and NaOH volumes in Direct method were 0.8, 1.0 and 1.2 mL.

Table 5 .
Results of recovery study by standard addition method *Mean value of three measurements